Colorectal carcinoma (CRC) is the third most common cancer in the United States, with an estimated 150,000 new cases occurring annually. Early detection of clinically relevant colorectal neoplasia (CRN), i.e., CRC and advanced colorectal adenomas (A-CRA), is the best way to improve survival. However, available screening modalities are impractical due to invasiveness and cost (i.e., colonoscopy) or insufficient diagnostic accuracy (i.e., fecal-based blood tests). A better approach to screening and surveillance would be highly desirable. Abnormal DNA methylation is an important epigenetic mechanism that has been strongly implicated in the pathogenesis and progression of colorectal neoplasia. Aberrant DNA methylation is detectable in serum making it an ideal candidate biomarker species for non-invasive screening. Previous studies have achieved limited success in developing definitive sets of methylation biomarkers for CRC screening due non-comprehensive approaches, and a failure to include all CRNs as meaningful targets for clinical discovery. Also, cohorts used in serum-based studies have been insufficiently powered and lacked independent validation sets. In this proposal, innovative strategies will be applied to permit genome-scale methylation mapping using tissues and sera from patients with CRN, and compared with tissues and sera from individuals without CRN. A novel and powerful new approach is being proposed to identify new biomarkers and methylation targets with the highest sensitivity and specificity, and will be validated using large, well- characterized sample sets through the following Specific Aims. Aim 1: Candidate DNA methylation biomarkers will be discovered using next generation bisulfite sequencing in matched tissue and serum from patients with CRN and individuals with a normal colon. Aim 2: Candidate serum methylation biomarkers will be discovered that distinguish patients with CRN vs. those without CRN, and validated biomarkers using quantitative PCR assays. Aim 3: Clinical validation of prioritized serum methylation biomarkers identified in Aim 2b will be performed in asymptomatic average risk individuals. The innovation of this project is based upon the first use of a novel, next generation sequencing-based approach for identifying methylation-based biomarkers using a genome-scale assay for methylation sites linked to colorectal neoplasia, and validating these newly discovered biomarkers using a large, well-characterized cohort of patients with CRN and controls. The long-term goal and potential impact of this project is to develop a sensitive, specific, non-invasive and inexpensive diagnostic test for early colorectal neoplasia.